Re: [ccp4bb] PHASER MR solution
Goettingen GPG Key ID = A46BEE1A -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] ligand bonds (AlF3) breaking up after refinement in refmac
Dear Anusman - It actually looks like the refinement didn't go so badly (the atoms are all in electron density), it's just that the Al - F bond lengths are a bit too long for Coot to draw them as bonds. I don't know what the correct bond distances should be (consult the literature and the PDB for that), but if your refinement is not giving you those bond distances, perhaps your molecule and the dictionary restraints don't match. You should look at the section near the beginning of the Refmac log file where it mentions disulfide bonds and other non-standard bonds to be used. You're looking for the bonding statements referring to the atoms of your ligand, like this: $TEXT:Warning: $$ Potential and actual links $$ WARNING : link:NAG-ASN is found dist = 1.440 ideal_dist= 1.439 ch:AA res: 350 ASN at:ND2 .-Ab res: 601 NAG at:C1 . If the statement for your AlF3 says link is found (not be used) that means your link restraints won't be applied in the refinement. The usual result of this is for the atoms to drift apart from each other somewhat; this would give you the result you see. I hope this helps! - Matt On 1/16/15 2:07 PM, ansuman biswas wrote: Dear users, I have a data at 2.2 A resolution. I am able to model AlF3 into the electron density (fig attached). However after one cycle of refinement the AlF3 molecule is exploding and the atoms move apart (fig2). AlF3 is already present in refmac library. First, I used that. But it broke up after refinement. Then, I extracted AlF3 coordinate from already published PDB and prepared the cif file. But, it also failed. I modified the cif file by changing the bond lengths according to the broken AlF3 structure but it was of no help. Kindly suggest how to carry out the refinement. regards, Ansuman -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] how to measure the angle between two aromatic ring plane in PYMOL?
Hi Bing - You don't need a fancy program to get the angle. If plane_wizard gave you a vector normal to the ring planes, you can use that. Otherwise, calculate the cross product between two vectors in the plane (e.g. C1-C4 and C2-C5 in a six-membered ring). This vector is normal to the ring plane. Once you have those ring plane normal vectors, just calculate the dot product between the two vectors. The result will be the product of the two vector magnitudes, multiplied by the cosine of the angle between them. The angle you want is 180 degrees minus the angle you get here. OR, you could put both aromatic rings up on a graphics terminal, and adjust your view so both rings are edge-on (they look like lines on the screen). Then put a protractor up to the screen and measure the angle! :D Hope that helps, Matt On 9/25/14 5:28 PM, Wang, Bing wrote: Hi everyone, I want to tell an angel between two aromatic planes which comes from two different molecules and cross each other. First, I tried the easier ways. I presume these two aromatic rings are in the perfect planes, that is why I tried plane_wizard.py and draw_plane_cgo.py which work very well. However after I got two plates by plane_wizard or draw_plane, I don't know how to measure the angle between these two plates. I also don't know whether I could get the angle I wanted from these two ways. I tried vector_angly.py which cann't loaded into pymol properly. If it could, how can i do to get the angel from these two plates I drew. Solutions? Since I am in the beginner state, please show me details which i could follow step by step. Second, If these two aromatic rings are not in the perfect planes, how can i find the best fit planes? And then find the angle between the best fit planes? I tried svdplos.py and makeCGOplates.py which are downloaded on line. unfortunately both of these can't be loaded into pymol properly. Solutions? Thanks! Bing Wang -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] rigaku micromax 007 cooling
Hi Andreas - My 007HF is cooled by a Haskris R050 chiller which I got from Rigaku. This has a refrigeration loop inside it to extract heat from the closed water loop that goes to the generator; the extracted heat can then go to building chilled water, or even an open loop (city cold water, then down the drain) if your building codes allow it. The advantage of this system is that it can handle unreliable chilled water temperature and flow, with allowable source water temperatures as high as 85 F. If your building water is really unreliable, you can even get an air-cooled Haskris - although your room A/C has to be able to handle the heat load in that case. Contact me off-list if you want more information. The system has been really reliable for me. - Matt On 7/24/14 11:53 AM, Andreas Förster wrote: Dear all, I have a Rigaku MicroMax 007 HF X-ray generator that is cooled by chilled water supplied rather unreliably through the building infrastructure. I was wondering what alternatives exist. Could other MicroMax 007 users share their experiences with alternative cooling solutions with me? Thank you. Andreas -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] emergency substitute for RT loop cover?
Hi Frank - How about a gel loading pipet tip as a substitute for the quartz capillary? Suck the crystal into it, then try to get it to stick to the wall. Flame seal the tip end, and use a glob of vacuum grease for the other end (or cut off the skinny part with your crystal using a razor blade). That semi-transparent plastic FPLC tubing (Tefzel?) might work as a substitute for the Mitegen capillary sleeve. Your Xray absorption and background scattering will be really high from all this plastic, but any port in a storm. - Matt On 7/7/14 12:32 PM, Frank von Delft wrote: Hi all Pretend you were stuck having to do RT data collection but without access to either Mitegen MicroRT Capillaries or the more old-fashioned quartz capillaries, to pop over the loop. Anybody have suggestions of alternative ways of doing this? I do want to use loops (I never learnt how to suck up crystals in capillaries). I have access to a passably stocked biochemistry teaching lab, and could at a pinch go rifle some more advanced research labs. (No, I'm not at home ;) Thanks! phx -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] Crystals Disappearing Overnight
Hi Maria - I think you already have a likely answer, but here's another one which I have observed many times: Temperature differences. If your crystallization plate is moved from one temperature to another (lab vs. crystal incubator), or even if it's left on the bench but the lab temperature varies overnight, you can get a temperature difference between your reservoir solution and the rest of the crystallization chamber. This can cause water condensation on the cover slip, which will dilute a hanging drop (and even a sitting drop if it's high enough up) and cause things to dissolve. The solution to this one is just careful control of the crystal plate environment. The same is true after the crystals are done growing - you can lose an entire drop of nice crystals from taking the plate out of the incubator to look at it under the microscope! Hope that helps, Matt On 5/2/14 7:39 AM, dusky dew wrote: Dear All, I am trying to crystallize a protein with Adenosine. My protein is in 20 mM Tris, 300 mM NaCl and the crystals appear in a condition with 5 percent PEG8K, 0.1 M Sodium Cacodylate. The protein is incubated with adenosine for 1/2 hr before setting the drop. The crystals appear right after the drop is set but unfortunately they dissolve overnight. The plate is kept at 16 degree. Could anyone elaborate on this. Is it possibly occurring because Adenosine has stability issues. Thanks for your suggestions. ~ Maria -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] AW: [ccp4bb] relation between redundancy and total reflection
Hi Faisal - These numbers can be found near the bottom of the Scalepack log file. In HKL2000, this has a default name of scale.log but you can rename it to anything you want in the Scaling window of HKL2000. You will find a table that looks like this: Summary of reflection intensities and R-factors by batch number All dataLinear Batch # obs # obs 1 I/sigma N. Chi**2 R-fac 1 873 873 5.7 2.0520.184 2 1551 1551 5.3 1.7630.178 3 1499 1499 6.0 1.5820.147 . . . . . All films 1072444 1072075 6.0 1.4280.157 The number 1072444 represents the total number of observations in this data set. Directly below this table is one titled Summary of observation redundancies by shells. The bottom line of that table looks like this: All hkl253 369 438 502 626 1647 2351 8257 24834 22607 61631 The number 61631 is the number of unique observations. Divide 1072444 by 61631 and you get 17.4, the overall redundancy for this dataset. (I was trying to use high redundancy to squeeze a bit more resolution out of a weakly diffracting crystal.) In recent versions of HKL, the redundancy values are printed in a table as well. Hope that helps, Matt On 4/17/14 10:25 AM, Faisal Tarique wrote: Dear Herman Where these values can be located..i.e. total no of reflections and no of unique reflections..which processed log file is the optimum one to look into..?? regards Faisal On Thu, Apr 17, 2014 at 7:48 PM, herman.schreu...@sanofi.com mailto:herman.schreu...@sanofi.com wrote: Dear Faisal, redundancy is total no. of observed reflections divided by no. of unique reflections, i.e. how often each unique reflection has been measured on average. Herman *Von:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Faisal Tarique *Gesendet:* Donnerstag, 17. April 2014 16:12 *An:* CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK *Betreff:* [ccp4bb] relation between redundancy and total reflection Dear all Can anybody please tell me how redundancy is related to total no. of observations and number of unique observations..what is the best way to identify and locate these values in a data processed through HKl2000..I know that completeness, redundancy, Rsymm, I/isig etc can easily be located in the log file but i am more concerned about locating of total reflections and no of unique reflections and its relation to redundancy.. -- Regards Faisal School of Life Sciences JNU -- Regards Faisal School of Life Sciences JNU -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] small molecule crystallography
Hi Andreas - My two cents, from having tried this a couple of times on small molecule crystals that crossed my path: - You can get an answer using only standard macromolecular crystallography programs. It may not be up to the standards of the small molecule community, but that's probably not what your student needs. - Denzo can handle small molecule diffraction - the key is to collect enough spots on each image. Use big oscillation ranges (10-20 degrees) and push the detector in as far as possible, with 2theta offset as well. - Use very short exposure times to avoid overloads. I also turned my generator down to minimum power. If your optics permit it, you might also slit down the beam. - Even with small molecules, crystal quality is important. Make sure you have good-looking diffraction - no streaky spots, multiple lattices, etc. The great thing about small molecule crystals is that you can always break off small pieces if the big chunk isn't clean enough - you have diffraction intensity to spare! - If your molecule of interest is chiral, you won't have to worry about the centric space groups that protein crystallographers never think about. If it's not (or there's a chance of a racemic mixture), then you should probably process in P1 and use Pointless to tell you the space group. - You can use SHELXS for direct methods structure determination if you're able to collect high enough resolution data. I believe this should work with 1.2 A data, and it ought to work well with 1.0 A or better. I found these webpages useful in helping me interpret the SHELX output: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Solve_a_small-molecule_structure http://shelx.uni-ac.gwdg.de/tutorial/english/shelxs.htm - If you have a heavy atom (iodine, iron, etc.) in the small molecule, you can probably get a phasing solution by SAD even with Cu Kalpha. You could probably even solve it by looking at the Patterson maps! Good luck! If it works, I think you'll be done in an afternoon. If it doesn't work, I'm not sure how to troubleshoot things. - Matt On 3/24/14 2:04 PM, Andreas Förster wrote: Dear all, I've been approached by a materials student with a petri dish full of big, sturdy, salty, yellow crystals. He claims I have the best kit for single-crystal diffraction on campus. I would very much appreciate advice on how to deal with this, anything in the range from won't work to use software X to analyze data in space group P-43N would be welcome. Thanks. Andreas -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] Table in NSMB
Hi Jan - I believe that ligand in this case refers to small molecule ligands, e.g. ATP or NADPH. If your ligand is a protein or even a peptide, I think it belongs in the protein category. (Thus, you may have no ligand atoms, unless you have bound sulfate, PEG, etc.) - Matt On 2/18/14 11:19 AM, Jan van Agthoven wrote: Dear all, I'm filling out my table for NSMB, about a structure of protein ligand bound to a receptor. They ask for 3 different lines regarding number of atoms bfactor. 1) Protein 2) Ligand/Ion 3) Water. Does my protein ligand belong to Protein or Ligand/Ion? Thanks, -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] no link_id
Hi Ronnie - Refmac has done the job for you (or for your friend). The Refmac log file says that Refmac has created a file called .cif (the exact name varies). You should open up that file and inspect it; it will be Refmac's best guess about the geometry of that link. I usually specify only bond lengths and bond angles, leaving torsion angles undefined (i.e. deleting those restraints) unless I know it should be restrained to a certain angle. The next time you run Refmac, use the Library file input line in ccp4i to feed it this cif file; refinement should proceed OK after this. You can also append the contents of the cif file that Refmac made to your drug.cif file. Just cut and paste with a text editor. Don't forget that you need to give your ligand a real 3-letter code, not just UNK, before depositing it. So you might as well do it now. (Even if the structure will remain private, not deposited, I would still change the code.) - Matt On 2/5/14 10:59 AM, Ronnie wrote: I am asking this question on behalf of a friend: -Thanks! Hi All - I am working at small biotech. We have minimal budget for software, only CCP4 and Coot. We have a ligand that binds covalently to cysteine. I define the parameter file using Prodrg in CCP4 to get drug.cif file and then make a link in Coot between the Cys and the Drug. The problem is Refmac keeps complaining that there is no link_id for this bond: I am reading library. Please wait. mon_lib.cif WARNING : link: CYS--UNK is without link_id link will be created with covalent bond only. Bond= 1.610 WARNING : description of link:CYS-UNK is not in the dictionary link will be created with bond_length = 1.610 Where do I specify this link_id, and where can I find an example for the correct syntax? Is this done in mon_lib.cif or the drug.cif file?? The bond length, if I'm not mistaken should be ~1.82 A for distance between carbon and sulfur. Thanks for any help. -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] off-topic: bug busting
Hi Phoebe - Cost-effective may not be the applicable word here, but the Microfluidizer works very well: http://www.microfluidicscorp.com/index.php?option=com_contentview=articleid=19Itemid=76 This gadget runs on house compressed air (don't try to use a compressed air tank - you'll empty it in minutes). It's a bit noisy, but so is a sonicator. The Microfluidizer really shines with large volumes of lysate - like 1 L and up. If you're only processing 100-200 mL at a time, I think sonication is the best way to go. Hope that helps, Matt On 2/4/14 11:49 AM, Phoebe A. Rice wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu mailto:pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] making high res image in pymol
Hi Alex - I'm not surprised you're having memory issues. I just tried this test with one of my molecules. Simply generating and displaying the symmetry mates in a 250 A radius required 4 G of memory, and the raytracing would have needed a lot more (I only have 8 G on this computer, so I didn't finish that job). Unless you're running your job on a server-scale computer with 32 G or more of memory, I think you'll have trouble with so many molecules. However, that's with cartoon rendering. I assume you're trying to show unit cell packing, and the molecules will be so tiny that it doesn't matter what they look like. Use ribbon rendering instead, and the memory demands are greatly reduced. I was able to raytrace a 2000 x 2000 pixel image (which should be more than adequate for a poster - that's 10 x 10 inches at 200 dpi) in 40 seconds with peak memory usage under 2 G. This is with the Fink compiled version of Pymol - version 1.6.9.0. Hope that helps, Matt On 1/28/14 10:27 AM, A K wrote: Hi all, I am trying to generate a high resolution figure of a molecule together with its symmetry mates (250 A readius) for a poster. If I try to ray it, the pymol session crashes (perhaps too many molecules are open). Using png xxx.png, dpi=300 or dpi=600 command doesn't make any difference; the image is still kind of low resolution for A0 or A1. Any idea how I can generate this in pymol? (I am using the free version of pymol) Thank you in advance, Alex -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] Two P1 xtals with same xtal contacts give 2 different asymmetric units
Dear Gabriel - You don't say if the two crystals are essentially identical - do they have the same unit cell parameters? I would imagine so. The phenomenon you describe is very common with molecular replacement (is this how you solved it?) or with auto-tracing. Your two structures are, in fact, identical within the limits of error of the refinement - this is why they give the same R values. There is no crystallographic difference between monomer A and monomer A' which is related by a crystal symmetry transformation. Either one (but not both!) can be included in the asymmetric unit, but we generally pick a set of monomers that gives a compact, pleasing arrangement. I expect that both of your arrangements can be reorganized to give two complete tetramers, by taking the orphan monomers and applying crystal symmetry transformations (simple unit cell translations, for P1) to place them in the equivalent position that completes the desired tetramer. Coot (and most other graphics programs) will generate the symmetry-equivalent molecules for you and allow you to save them, then you can just extract chain A (for example) from one PDB file, and paste it into the other PDB file in place of the chain A that's already there. Feel free to contact me if that explanation wasn't clear. - Matt On 1/22/14 3:50 PM, Gabriel Moreno wrote: Dear CCP4 Contributors, I have a bit of a mystery: Two co-crystals that I picked up from the same grid tray (the two conditions vary slightly in %PEG and [salt], both indexed as P1 (apo structure normally crystallizes in P3221). One dataset was indexed, integrated and scaled with HKL2000. The other was processed with MOSFILM (could not process in HKL2000). Downstream processing for both sets was done exactly the same in PHENIX. Though both asymmetric units contain two complete tetramers, the interesting thing is that the configuration of monomers is different between the solutions. One contains one complete tetramer, one trimer (with a void where the fourth monomer would be), and one monomer on off on its own. The asymmetric unit of the other dataset solution also contains a complete tetramer, but then has two dimers. Close analysis of contacts between symmetrically related molecules reveals that the crystal packing is exactly the same between the two solutions from the two datasets. Also, the Rwork and Rfree for both models are 0.18 and 0.20. Other quality indices are also comparable between the two sets. Here's my question: Does this phenomenon reveal anything important, or is this type of thing just seen sometimes with P1 solutions from crystals of the same protein and condition (more or less). I hope I have been clear. Thanks! Gabriel -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] Protein concentration form chromatograms
Hi Karel - To add to what Jurgen said, a few points on the measurement of the protein peak from the chromatogram. - I usually approximate the peak as a triangle, so that the total peak area is 1/2 height (absorbance maximum) x base (the number of ml in your pool) - If your peak is a strong one, watch out for non-linearity in the absorbance measurement - my Akta UV monitor doesn't give a reliable reading once the A280 goes above 1.8. This will cause you to underestimate your total protein amount. - You also need to apply a correction factor since your UV cell path length isn't 1 cm. You could look up what the path length is in the manual, but the easiest way to do this is to compare UV readings for a set of fractions from your FPLC monitor and a standard spectrophotometer. Figure out the ratio of the two (it'll be a simple whole number, probably 2 or 5, or maybe 1 if your UV monitor does the correction automatically), then put it on a post-it next to the UV monitor so you won't have to do this again. Now multiply your chromatogram's integrated peak area by this factor to give you the standard (1 cm path length) A280 measurement. Hope that helps, Matt On 1/15/14 10:09 AM, Karel Chaz wrote: Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] Best compounds for heavy atom soaks
Hi Rhys - Don't forget to try sulfur-SAD, especially the multi-crystal version published recently: http://journals.iucr.org/d/issues/2013/07/00/ba5189/index.html This seems well suited to your situation. - Matt On 1/15/14 12:18 PM, RHYS GRINTER wrote: Hello message board, My group has some crystals of an interesting protein to take to the synchrotron in a couple of weeks. We won't be able to prepare and crystallise a SelMet derivative during that time period, but we have loads of crystals sitting around. The diffraction isn't great, we see maybe 3.5 at home but might be enough to get over the line. It will be a very difficult MR target, so we were thinking of soaking so crystals with heavy atomic compounds that we have lying around. I was wondering if people had any suggestions of compounds that people have used successfully for experimental phasing and maybe concentrations to use and soaking time. Cheers, Rhys -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] Fix cell dimensions
Hi Niu - In HKL2000, you should first get an indexing solution with the 40 A axis, as you have done previously. Then go to the tab of the GUI labeled Macros. In the bottom row, Immediate Execution, you enter the following: unit cell a b c alpha beta gamma where you replace the words after unit cell with the desired unit cell dimensions, including your 20 A axis. (Make sure you use exactly half of the previous cell dimension.) Then hit Run Macro. If it works correctly, you should see every other predicted spot disappear in your XdisplayF window. Now look to see if those predicted spots should have been removed or not! Use the zoom window to help with this. You should be able to refine the new cell parameters normally. Hope that helps, Matt On 11/18/13 5:03 PM, Niu Tou wrote: Dear Andrew, As previously I posted a MR case which has a significant 95% off origin peak, some experts suggested to reprocess the data with cutting one axis to half, from 40A to 20A. I tried HKL2000 and XDS, none of them is willing to give a solution with 20A, even I specify it in XDS script. So I wonder is there any way to force this work to be done. Thanks! Best, Niu On Mon, Nov 18, 2013 at 4:56 PM, Andrew Leslie and...@mrc-lmb.cam.ac.uk mailto:and...@mrc-lmb.cam.ac.uk wrote: Dear Niu, It depends on which part of processing you are referring to, i.e. the indexing step or the integration step. In MOSFLM there is no way to enforce cell dimensions during indexing, but providing there is an indexing solution that has cell dimensions close to the ones you want, you can enforce a (slightly) different set of cell dimensions during the integration step. Normally other refined parameters will ensure that you still get a good prediction of spot positions. I suspect that this can be done in other programs too. Without knowing why you want to do this, I cannot comment on whether this is the best procedure to follow. Best wishes, Andrew On 18 Nov 2013, at 21:48, Niu Tou niutou2...@gmail.com mailto:niutou2...@gmail.com wrote: Dear All, Does any one know how to strictly fix the cell dimensions during data processing? In HKL2000 there is only a keyword to define the longest vector. In XDS there is a option to input cell parameters, but sometimes the program would not follow the input values and switch back to the one it thinks best. Any suggestions will be appreciated. Thanks! Best, Niu -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] observed criterion sigma
Dear Faisal - HKL2000 (Denzo/Scalepack) use I greater than -3 sigma (that's NEGATIVE 3) as the observed criterion, so that's what you would put down for this entry. There is another place where you're asked to provide an observed criterion for F's used during refinement. I always put down 0 (i.e. use all F's) for this one. I have no idea what Scala does. - Matt On 11/14/13 6:06 AM, Faisal Tarique wrote: Dear all I request you to please explain the Observed criterion sigma value required during pdb deposition. Does it depends on the methods of data integration and scaling ? if yes then what are the values for the data processed through scalepack2mtz (HKL2000) and scala (mosflm).. -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] SA-omit map
Hi Deng - Can you tell why the reviewer was asking for the SA-omit map? Is there some doubt about the conformation of your ssDNA, or even whether it is present in the first place? Is there a question about the sequence, or the sequence register (which nucleotides go in which positions)? Even a poor-quality map should let you locate the phosphate backbone of the DNA. This should convince most reviewers that the DNA is present and in the general position you have modeled. You can't resolve concerns about nucleotide base position or conformation without a better quality map. I imagine that you built the protein model first during your structure determination, and only added the ssDNA later in the refinement. If this is correct, you could present the maps from the last refinement cycle before you added the DNA. This is even less model biased than what the reviewer wants. Hope that helps, Matt On 11/4/13 1:36 AM, dengzq1987 wrote: Dear all, Recently, I received the comments from referees, they asked for the SA-omit map of the ssDNA of our protein-DNA complex. They said that simulated annealing omit map better than a biased 2Fo-Fc. The ssDNA consists of seven thymidine nucleotide. Our data diffracted to 2.65A,but the data quality is not good and twin. We tried to produce SA-omit map using phenix. The map is really bad. Does anyone have suggestion to refine the map? Thank you! Bests, zq Deng 2013-11-04 dengzq1987 -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] A photograph of the Arndt-Wonacott rotation camera?
Dear Gerard - Here is one picture, but I'm afraid it's quite small and not good quality: http://books.google.com/books?id=S9PPHvJ8otMCpg=PA28 I assume the camera is that object with the multiple circles in the left center of panel A? Also, these folks seem to have one in their collection: http://collectionsonline.nmsi.ac.uk/detail.php?t=objectstype=allf=s=arndt+wonacottrecord=0 No image that I could find online, but perhaps they would share one with you? Hope that helps, Matt On 10/30/13 12:05 PM, Gerard Bricogne wrote: Dear all, Apologies for such a retro and non-biological question, but would anyone have a photograph of an Arndt-Wonacott rotation camera that he/she would be willing to share? I collected data on the first two prototypes in the early seventies, then on one of the first commercial models, but I cannot find any images of this ground-breaking piece of equipement on the Web. I found images for the Enraf-Nonius precession camera and the CAD-4 diffractometer, but not for the A-W rotation camera. This would be for use as visual material in presentations, and I would gratefully acknowledge the source of it. Thank you in advance! With fingers crossed ... . Gerard. -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] changes in small sections of secondary structure
Dear Mahesh - I can't comment on the energy changes for the protein, but I can say that the changes you've highlighted are not really due to alterations of the protein, but rather inconsistency in assignment of secondary structure. If you look closely at the areas you've highlighted, you will see that the C-alpha backbone as depicted in this cartoon rendering is still making a helix even in the structures where it is not drawn as an alpha-helix. This typically means that some residues have just fallen below whatever threshold the rendering program uses to define secondary structure (like position on a Ramachandran plot) in one structure, and the same residues are just above the threshold in the other structure. This is not a significant change. Indeed, different programs may render the same structure differently. I hope that clears up some confusion. All the best, Matt On 10/18/13 2:52 PM, Mahesh Lingaraju wrote: Hello experts Attached is an image showing different crystal structures of the same protein in diffrent states (mutant vs substrate bound vs unliganded) and highlighted are little changes in secondary structure. I was wondering how real such small changes are and if they are real could they be enough to perturb the energy potential of the protein significantly. I apologize if this is a naive question as this is clearly not my area of expertise. Thanks for your input Mahesh -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] Supplier for X-ray sensitive paper
Hi Joe - This is what I've used in the past. It's film, not paper, but needs no developer and is instant-readout. I think the find a distributor link should help you obtain it. http://www.ashland.com/products/gafchromic-radiology-films - Matt On 6/26/13 3:51 PM, Patel, Joe wrote: Hi All, I have done a few searches of the archive and googled a few times but not found what I am looking for. Could someone point me in the direction of a supplier of the X-ray sensitive paper I have used in the past to confirm beam position on a home source. I am specifically after this type of paper rather than X-ray film so as not to have to go through any developing stage and quickly visualize the location of the beam at different points beyond the position of the goniometer towards the detector. A USA supplier would be great but any would do. Many thanks, Joe P -- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] Puzzling observation about size exclusion chromatography
Hi Zhen - Superdex is known to have some ion-exchange characteristics, so that it can weakly interact with some proteins. This is why the manufacturer recommends including a certain amount of salt in the running buffer. I have had the same experience with a few proteins, including one that came off the column well after the salt peak! (The protein was very clean after this step; all other proteins had eluted earlier.) As others have said, you can't rely on molecular weight calibrations in this case, but this behavior alone is no reason to think that the protein is misfolded or otherwise badly behaved. If you don't like the late elution, try increasing the salt concentration of your running buffer to 250 or even 500 mM. You'll probably need to exchange the eluted protein back into a low-salt buffer for your next steps (e.g. crystallization) if you do this. - Matt On 6/20/13 3:09 PM, Zhang, Zhen wrote: Dear all, I just observed a puzzling phenomenon when purifying a refolded protein with size exclusion chromatography. The protein was solubilized by 8M Urea and refolded by dialysis against 500mM Arginine in PBS. The protein is 40KDal and is expected to be a trimer. The puzzling part is the protein after refolding always eluted at 18ml from the superdex S200 column (10/300), which is calculated to be 5KDal by standard. However, the fractions appear to be at 40KDal with SDS PAGE and the protein is functional in term of in vitro binding to the protein-specific monoclonal antibody. I could not explain the observation and I am wondering if anyone has the similar experience or has an opinion on this. Any comments are welcome. Thanks. Zhen The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail. -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] Crystallisation below 0°C
Hi Glenn - I have nothing systematic, but I remember that transducin-alpha (1TND) was crystallized at -12 C, with 20% glycerol in the mother liquor. (The crystals grew at higher temperatures, but weren't as good.) I worked on this project briefly in grad school, and I remember that looking at the crystals was a big nuisance - you had to take the trays out of the -12 freezer, run to the cold room, and look at them quickly before they warmed up too much! - Matt On 5/30/13 7:26 AM, Glenn Masson wrote: Hello CCP4BBers, I am currently playing with some crystals that seem to enjoy lower temperatures, and I was thinking of breaking the 0°C threshold. Looking for examples of this in the literature is problematic, as searching for examples in the PDB (Under the advanced search- crystal properties- temperature (K)) turns up a large amount of false positives. Many otherwise supremely intelligent people seem unable or unwilling to grasp the concept of Kelvin (it's amazing how many protein structures were solved only 22 degrees above absolute zero...). I was wondering if anyone has much experience in this area? I see a few structures e.g. 2Z97 (-5°C) and 4H0W (-2°C), but I was wondering if anyone has a more systematic knowledge, some more examples, and what the parameters and best practice of this technique are. Many thanks, Glenn Masson MRC-Laboratory of Molecular Biology -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] OFF TOPIC
Hi Rex - You can usually get the three-letter code from the PDB itself. At top of the PDB home page, you can see that one of the options for the search bar is Ligand. If you put in a (mostly) correct name for the ligand, you will get back the three-letter code that the PDB is using. This should match exactly with the Refmac ligand dictionary that Joel mentioned. Another place to look for ligand names is Gerard Kleywegt's HIC-UP site ( http://xray.bmc.uu.se/hicup/ ). I find that their search function isn't so great, so I get the entire 7870-compound list by name here: http://xray.bmc.uu.se/hicup/xname.html and do a text search for something resembling my compound. You can then download coordinates (real and idealized), ligand dictionaries, etc. Furthermore, if the compound you're working with isn't in this list, it's new to the PDB, and you get to give it your own three-letter code! (All the good ones are already taken, unfortunately.) Hope that helps, Matt On 10/7/12 5:43 PM, Joel Tyndall wrote: Hi Rex, In Coot, under the file menu, “Get Fragment” and the type LBT (upper case). This (LBT) is the three letter code for alpha-lactose. Unfortunately it is not that easy to find the codes. However on my windows machine the list is here: C:\CCP4\6.3\lib\data\monomers\list Hope this helps Joel *From:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Rex Palmer *Sent:* Monday, 8 October 2012 9:12 a.m. *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] OFF TOPIC Can anyone please tell me how to extract the pdb for a ligand, eg alpha lactose, from the COOT library, prior to fitting into difference density. Thanks in advance. Rex Palmer http://www.bbk.ac.uk/biology/our-staff/emeritus-staff http://rexpalmer2010.homestead.com -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] ideal rms bond length
Dear Faisal - There are a few problems with your refinement. First and foremost, your R factors increase instead of decreasing! This is associated with a decrease in geometry rmsd, suggesting that Refmac is trying to fix an incorrect model using an excessively tight geometry weight. Second, your geometry rmsd values are still quite high. You don't say what resolution you have to work with, but given your R factors, I would expect you have something in the 2 to 2.5 A range. Your rmsBOND values would typically be below 0.015 at this resolution. It's possible you have a few areas of your model with very bad geometry which are raising the global values even though everything else is mostly OK. Third, I wonder why your R and Rfree are so close at the start of refinement. The low R values suggest a late-stage refined model, not an initial molecular replacement solution or something similar. If you're starting with an isomorphous structure, you need to use the same free R set as was used in the previous refinement in order to keep your R free set uncontaminated. You should examine your model carefully and look for regions that don't have strong electron density to support them. Also look for bad geometry: bond and angle deviations greater than 5 sigma (these will be in the Refmac log file), bad rotamers, bad Ramachandran position, etc. Regions which are of poor quality should be removed from the model and rebuilt into unbiased density in later refinement rounds. (Or if the electron density stays poor, leave the region out of the model.) Good luck, Matt On 10/2/12 5:49 PM, Faisal Tarique wrote: Dear all i request you to please answer my basic query about the ideal acceptable rmsbond length obtained during refmac refinement..is the data acceptable in mine case which is as follows.. NcycRfactRfree FOM -LL -LLfree rmsBOND zBOND rmsANGL zANGL rmsCHIRAL $$ $$ 0 0.2090 0.2079 0.875226315. 11985.5 0.0278 1.389 2.718 1.261 0.198 1 0.2064 0.2284 0.850226313. 12201.1 0.0285 1.427 2.733 1.271 0.204 2 0.2076 0.2373 0.837226944. 12289.9 0.0248 1.242 2.598 1.200 0.187 3 0.2092 0.2429 0.828227495. 12341.7 0.0222 1.107 2.458 1.128 0.173 4 0.2100 0.2468 0.822227753. 12372.4 0.0211 1.053 2.377 1.086 0.166 5 0.2104 0.2500 0.818227942. 12395.7 0.0204 1.021 2.326 1.061 0.161 6 0.2108 0.2522 0.814228075. 12411.5 0.0200 0.999 2.289 1.042 0.158 7 0.2111 0.2537 0.812228162. 12421.8 0.0197 0.984 2.265 1.030 0.156 8 0.2113 0.2550 0.810228228. 12430.5 0.0194 0.971 2.243 1.020 0.154 9 0.2114 0.2559 0.809228300. 12436.1 0.0192 0.962 2.228 1.012 0.153 10 0.2116 0.2568 0.808228348. 12441.7 0.0191 0.957 2.218 1.008 0.152 11 0.2118 0.2574 0.807228394. 12446.2 0.0190 0.951 2.210 1.004 0.151 12 0.2119 0.2581 0.806228421. 12449.6 0.0189 0.948 2.203 1.001 0.151 13 0.2119 0.2585 0.805228440. 12452.7 0.0189 0.944 2.198 0.998 0.150 14 0.2120 0.2590 0.805228461. 12455.0 0.0188 0.941 2.194 0.996 0.150 15 0.2121 0.2593 0.804228480. 12456.9 0.0188 0.939 2.190 0.995 0.150 -- Regards Faisal School of Life Sciences JNU -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] poorly diffracting and twinned trigonal crystal
improved the Rfree to 53.0. Refining with torsion angle dynamics and restrained group B-factor refinement again made the Rfree worse (increased to 54.5%). I analyzed the reflection file processed in P31 using detect_twinning.inp in cns. The data did not appear to be perfectly merohedrally twinned, but in the test for partial merohedral twinning, the twin fraction calculated for “2 along a,b” was 0.475. I repeated rigid body refinement, then torsion angle dynamics with restrained group b-factor using the calculated twinning parameters. This brought the free R down to 46.3%, but caused significant divergence between Rwork and Rfree (Rwork =21.4%(!)). The Rfree is fairly constant across resolution shells, but Rwork drops dramatically with low resolution reflections (In the 50 – 9.14 ang shell, Rwork = 12.3%!). I’m guessing that because the twinning fraction is near 0.5, detwinning is not working. Does anyone have any suggestions about how to successfully refine this structure (assuming it is possible)? Should we average the twin related reflections to generate perfectly twinned data, and if so, how do we do that? Is the twinning likely responsible for our difficulty refining the structure or could there be a problem with the space group assignment? Why does including the partial twinning in our refinement cause Rfree and Rwork to diverge so dramatically? Given the trouble I’ve had so far and the poor quality of the data, I’m about ready to give up on this structure, but if anyone has any ideas please let me know. -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] poorly diffracting and twinned trigonal crystal
Dear Ed - I agree with you, and I felt a little funny giving that advice, since I know that Rmerge is rather outdated as a resolution cutoff. What I was really reacting to was the slope of I versus resolution; if you look at Qing's Scalepack table, you can see that average I plateaus in the low 4 A range, and I usually get worried about the data when I don't see a steady decrease in I with increasing resolution. Having said that, I don't usually work with datasets at this resolution, and it's possible (even likely?) that I was fooled by the bump in the Wilson plot around 4. Perhaps this data does extend to 4.1. - Matt On 9/7/12 11:39 AM, Edwin Pozharski wrote: Matt, On 09/07/2012 09:56 AM, Matthew Franklin wrote: I'm also a bit dubious about the 4.3 A limit; your useful data may be ending around 4.6 instead, despite the high I/sigma numbers. Why? I would rather suggest Qing extends resolution to where I/sigma~1. Other than Rmerge, I don't see what else you may be looking at, and that is traditional but inferior way to determine resolution cutoff. Cheers, Ed. -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] Question about weird diffraction map
Hi Bernhard - Having the spots in regular lines indicates that this is a single crystal diffraction pattern (to a first approximation). I have seen ice rings which contain a few strong spots, probably from microcrystals of ice, but I haven't yet seen ice diffraction that shows a lattice of spots. So, the presence of the spot lattice, plus the spots below 3.9 A, allow one to say that this image isn't ice diffraction on top of weak/absent protein diffraction. Ergo, this crystal is not a macromolecule. Plus, I was trying to show Zhao that the spots aren't just scattered at random. - Matt On 7/23/12 5:25 PM, Bernhard Rupp (Hofkristallrat a.D.) wrote: you'll see that some of them are arranged in regular lines. I am not sure I understand what the line argument implies? indicates a very small unit cell, with dimensions probably 10 A Very indicative also the few strong and isolated high resolution reflections Cheers, BR -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] Question about weird diffraction map
Dear Zhao - This is a salt crystal. You do have spots, even if some of them are streaky, and if you look carefully, you'll see that some of them are arranged in regular lines. However, there are very few, and no spots below about 6 A. This indicates a very small unit cell, with dimensions probably 10 A, which can only be a salt or other small molecule crystal. The usual check for this is to rotate your crystal by 5-10 degrees, instead of the usual 0.5 - 1 degree. This will put more spots on the image, and it should become evident what you are dealing with. Consider all of the components of your protein buffer + crystallization solution - some combination (e.g. calcium plus phosphate) is most likely not very soluble, and can come out of solution. Hope that helps, Matt PS. Those crystal dyes are notorious for giving false positives. I never use them to check if a crystal is protein. On 7/23/12 4:52 PM, gengxiang zhao wrote: Dear CCP4BB, I have a question about my current diffraction map for one of crystals which can be found in the attachment. Basically, the crystals were dyed to initially testify that it belongs to protein. But from this diffraction, even in the low diffract angle, no diffraction spots theremeaning-this is not a protein crystal? Any experienced idea/questions welcomed to discuss here. Zhao, -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] help regarding structure solution
Dear Sonali - It seems very likely that your original protein (which did not crystallize) was proteolytically degraded over the one year storage, and you now have a fragment of the original protein which is capable of crystallizing. You should analyze all of the homologous structures in the PDB to see if there are break points, like domain boundaries, which could produce a fragment of the appropriate size upon cleavage. Then use those fragments as your search models. Don't forget to consider more distantly related proteins, or structures that match only a subdomain of your protein. However, it sounds as if you've already done that. Furthermore, if I read your email correctly, the original crystal is now gone, so there is no chance to collect additional data. (If this is not the case, you might attempt to get enough data for sulfur SAD phasing.) So what I suggest is that you try to reproduce the crystallization, not by waiting a year, but by limited proteolysis of the full-length protein. You should be able to get a stable fragment corresponding to the thing that's in your tray, and analyze it by mass spec and/or N-terminal sequencing. (You might be able to N-terminal sequence from your existing crystallization drop as well.) Once you know what the fragment is, you should try cloning and expressing just that fragment, then setting up more trays, perhaps with selenomethionine labeling. Good luck! - Matt On 6/20/12 2:13 PM, sonali dhindwal wrote: Dear All, I am working on a protein for last so many years and for which i have got crystal now in a tray which i kept 1 years ago. It diffracts well and resolution is 2.2A, which is good. I indexed in HKL2000, mosflm and automar and it shows P21 space group in all data reduction packages. But when I tried using molrep or phaser then I do not get any solution. The sequence of my protein is having 46% identity with other available crystal structure. Also when I tried to get matthews coffecient, it calculates its molecular mass less ( about 35 kDa) than which should be (original 54kDa) with solvent content 47%. I have also run the silver staining gel of the protein which contained crystal that shows about 45 kD protein band which is 10 less than the original. Also I tried to run gel on crystal but it did not give anything as it was a small crystal. I have tried all combinations of the search model and tried to break available pdb many ways to make different search models but have not got any good solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions. I will highly appreciate all the suggestions for this kind of problem. Thanks and regards -- Sonali -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] P21221 to P21212 conversion
On 5/7/12 4:09 PM, Shya Biswas wrote: Hi all, I was wondering if anyone knows how to convert the P21221 to P21212 spacegroup in HKL2000. I scaled the data set in P21212 in HKL 2000 but I got a correct MR solution in P21221 spacegroup. I have a script file that runs with scalepack but was wondering if there is an easier way to do it with HKL2000 gui mode. thanks, Shya Hi Shya - Under the Scale tab of HKL2000, you'll see a button near the bottom labeled Reindex. Clicking this brings up a dialog box with the reindexing conventions appropriate to your spacegroup. For P orthorhombic, there are only two choices: hkl - klh, or hkl - lhk. If you have P21221, and want to go to P21212, you want hkl -lhk. HOWEVER, there seems to be a bug in this particular reindexing (or maybe the options are written confusingly). You want to choose the wrong option (number 2 in the list presented), as the two options seem to be reversed. You'll know that you got it right by inspection of the scaling log file - look at the systematic absence table at the bottom. Also check that the unit cell axes were permuted in the correct way - you want your old b axis to be your new c axis. Once you select the correct reindexing (make sure you apply it to all datasets being scaled at the same time), you click Reindex in the popup dialog, and this triggers another round of scaling with the reindexing included. This reindexing is sticky - you don't need to select it again for subsequent rounds of scaling. Hope that helps - feel free to contact me if you want more explanation. - Matt -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] problem in scaling the Zn-MAD data
Hi - Let me just add that P312 is a very uncommon space group for protein crystals, much less common than P321. (This doesn't mean you don't have it - it's just unlikely.) If you look at PDB statistics: P 3 1 2 : 12 structures P3(1) 1 2: 61 structures P3(2) 1 2: 85 structures P 3 2 1 : 278 structures P3(1) 2 1: 2354 structures P3(2) 2 1: 2533 structures This also suggests, by the way, that you have a screw axis that you haven't accounted for yet. It won't affect your data scaling, but it sure will affect your molecular replacement job! Hope that helps, Matt On 4/5/12 12:31 PM, Deepthi wrote: Hello I arrived at the p312 space group by running a self rotation function using MOLREP. The maps show the space group as p312. I was scaling the data individually for each wavelength. None of the three wavelengths are scaling are scaling in p312 space group. On Thu, Apr 5, 2012 at 2:17 AM, Clemens Vonrhein vonrh...@globalphasing.com mailto:vonrh...@globalphasing.com wrote: Hi, On Wed, Apr 04, 2012 at 02:07:58PM -0700, Deepthi wrote: Hello everyone I have a problem scaling the MAD data which was collected a week ago.The data was collected at 1.5A resolution using three wavelengths for Zn-MAD experiments. Scaling the data for MAD experiments, the number of rejections and chi2 values were very high even after adjusting the error-scale factor and error model. The space group i used was p312 which i obtained by running a self-rotation function in MOLREP. When i scale my data using p312 spacegroup the chi2 and rejections were huge. But he data was scaling well in p321 spacegroup. can anyone explain whats going on? When you say 'Scaling the data for MAD experiments': do you mean scaling the various scans for your 3-wvl MAD data in a single scaling job? Unless you already took care of this during data integration, remember that your separate scans could have been indexed differently and therefore don't match up. See eg. http://www.ccp4.ac.uk/html/reindexing.html for some lookup-tables in P312 and P321. You can use the CCP4 program 'reindex' on MTZ files if needed. But I guess most modern data-processing and scaling programs will take care of that automatically anyway? Cheers Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) *** -- Deepthi -- Matthew Franklin, Ph. D. Senior Research Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (646) 275-7165
Re: [ccp4bb] REFMAC Riding Hydrogens
Hi Paul - Refmac does indeed add riding hydrogens by default; see here: http://www.ccp4.ac.uk/dist/html/refmac5/keywords/restraints.html#makecif The REMARK 3 is referring to what you think it is: riding hydrogens have been added. (They are not, however, written to the output file unless you request that.) I believe the H atoms are used for both stereochemistry and scattering, although there may be some fudging on the scattering factors. Adding the riding hydrogens generally gives you some improvement in R factors even with a good quality (i.e. stereochemically correct) model. If you look at the link I gave above, you will see that you can do three things with hydrogens: never use them, use the riding hydrogens, or only use H atoms given in the input file (which you could of course tailor to your needs). Hope that helps, Matt On 3/5/12 2:15 PM, Paul Smith wrote: Hello CCP4 community, I'm posting at large regarding a previously raised issue for REFMAC for which I cannot find the conclusion in the old threads. Specifically, does REFMAC add riding hydrogens during default refinement? Though I've not requested that any hydrogens be added, only used if in the input, I see the following in the output header: REMARK 3 HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS Does anyone know exactly what this remark is referring to? Are riding hydrogens added for stereochemical restraints, or are their scattering contributions calculated regardless of they are explicitly defined? Can the hydrogen behavior in REFMAC be more explicitly controlled. Thanks, --Paul -- Matthew Franklin, Ph. D. Senior Research Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (646) 275-7165
Re: [ccp4bb] Invisible Reference on Pubmed?
On 2/7/12 4:02 PM, Jacob Keller wrote: Dear CCP4BB, this is perhaps my most egregious off-topic post, but can anyone explain why the following reference is not findable in PubMed? I can get it from the ACS website, but not on PubMed or elsewhere. The journal is on PubMed--is it perhaps because it's funded by ExxonMobil? Very strange... Jacob Hi Jacob - PubMed isn't universal - its goal is to cover the biomedical literature, so articles on the margins, like organic chemistry, materials science (even crystallography!) aren't fully covered. If you look at PubMed's total coverage of the journal Macromolecules, you'll see that it's extremely patchy: lots of articles from 1977, then almost nothing until a whole fleet of articles in 1998, then almost nothing again until 2007. Your article must have fallen into one of these coverage holes. Either that, or it's THE MAN suppressing the research needed to cure cancer and the common cold, and build a car that runs on water... :) - Matt -- Matthew Franklin, Ph. D. Senior Research Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (646) 275-7165
Re: [ccp4bb] synchrotron X-ray picture
Hi Pat - I, too, have a memory of such a picture. This isn't quite the one I was thinking of, but it should hopefully serve the purpose: http://www.nsls.bnl.gov/about/imagelibrary/images/hr/Synchrotron_Light2_hires.tif (there are a couple more from the parent page: http://www.nsls.bnl.gov/about/imagelibrary/ ) - Matt On 2/1/12 1:43 PM, Patrick Loll wrote: Hi all, I have a vague memory of having a picture in someone's presentation once, showing a smoking hot X-ray beam emerging from the beam pipe in a hutch at a synchrotron. I think the picture might have been a double exposure, with a long exposure that captured air ionization superimposed on a normal photo (or some similar contrivance). In any case, can anyone point to or provide such a picture? I'm preparing a seminar, and I want to lend artistic verisimilitude to an otherwise bald and unconvincing narrative... Thanks in advance, Pat --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu -- Matthew Franklin, Ph. D. Senior Research Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (646) 275-7165
Re: [ccp4bb] Lithium versus Sodium
On 1/12/12 9:42 AM, Ed Pozharski wrote: On Thu, 2012-01-12 at 09:52 +, Patel, Joe wrote: Do you have ultra-high resolution? Something I did not…. Are there many examples in the pdb of proteins with Li+ refined? http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=n/atemplate=het2pdb.htmlparam1=_LI 39 in total. Some are fairly low resolution (2.8A), and only five are higher than 1.2A. I'd think that placing lithium ion should be based on some extra-crystallographic evidence, plus maybe some structural considerations such as correctly placed coordinating ligands. Since I'm responsible for eight of those structures, I'll just say that I thought fairly hard before building a lithium into that peak, knowing that I couldn't really distinguish it from water or sodium. I was working with a 1.7 A map, and I put the lithium there based on three criteria: - the crystals grew in something like 2 M lithium sulfate, whereas the only source of sodium would have been from the buffer or the protein solution - there were two negatively charged residues coordinating the peak in question, suggesting it was a cation - the bond distances were consistent with lithium coordination, for what that's worth at this resolution That was the first structure (1TW7), and all of the others were treated the same since it was the same crystals soaked with different compounds in the same conditions. - Matt -- Matthew Franklin, Ph. D. Senior Research Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (646) 275-7165
Re: [ccp4bb] Lithium versus Sodium
Hi Ed - There was a peak in the difference maps, as I recall. I believe it initially got built as a water, but that proved to be too many electrons, giving a negative peak. I removed the water, but it was clear that something needed to be there, at which point I started casting about for alternative atoms, and settled on lithium. I'm pretty sure I never tried to put sodium in there and see if it refined. Caveat: this work was all done eight years ago, and I don't have access to any of the files anymore. So I can't verify any of these statements! However, I did deposit Fobs for these structures, if you care to make your own maps... I just checked the structure in EDS, and the peak for the lithium is pretty low, around 0.6 sigma. I would say that it looked better in the maps I was using. Hope that helps, Matt On 1/12/12 11:22 AM, Ed Pozharski wrote: Matt, thank you, this is an excellent summary. One question remains - the lithium peak should be, afaiu, much lower than the water/sodium. Was there a peak in difference map or was placement based on identifying something that looked like a coordination site? Cheers, Ed. On Thu, 2012-01-12 at 10:23 -0500, Matthew Franklin wrote: On 1/12/12 9:42 AM, Ed Pozharski wrote: On Thu, 2012-01-12 at 09:52 +, Patel, Joe wrote: Do you have ultra-high resolution? Something I did not…. Are there many examples in the pdb of proteins with Li+ refined? http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=n/atemplate=het2pdb.htmlparam1=_LI 39 in total. Some are fairly low resolution (2.8A), and only five are higher than 1.2A. I'd think that placing lithium ion should be based on some extra-crystallographic evidence, plus maybe some structural considerations such as correctly placed coordinating ligands. Since I'm responsible for eight of those structures, I'll just say that I thought fairly hard before building a lithium into that peak, knowing that I couldn't really distinguish it from water or sodium. I was working with a 1.7 A map, and I put the lithium there based on three criteria: - the crystals grew in something like 2 M lithium sulfate, whereas the only source of sodium would have been from the buffer or the protein solution - there were two negatively charged residues coordinating the peak in question, suggesting it was a cation - the bond distances were consistent with lithium coordination, for what that's worth at this resolution That was the first structure (1TW7), and all of the others were treated the same since it was the same crystals soaked with different compounds in the same conditions. - Matt -- Matthew Franklin, Ph. D. Senior Research Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (646) 275-7165
Re: [ccp4bb] refmac problem with anisotropic Us
On 6/10/11 6:10 PM, Ed Pozharski wrote: On Fri, 2011-06-10 at 12:48 -0700, Ethan Merritt wrote: I don't think that combination makes any sense. Whatever anisotropic is being described by the TLS parameters can also be fully described by the individual anisotropic U^ij terms. So the TLS parameters are entirely redundant, leading the minimization function to be poorly defined. True, but the TLS refinement is implemented in refmac as separate step preceding the positional/adp refinement. So in theory the TLS step will go fine, and the anisotropic ADP refinement will take care of anisotropy additional to TLS component. But of course you are absolutely right that I am virtually certain that refinement of individual anisotropic U^ij terms cannot be justified at 1.8A. Too many parameters, too few observations. Matt reports that TLS lowers R/Rfree compared to anisotropic ADPs alone. Well that is good (TLS works!), but the question is if the TLS +aniso is better than TLS+iso or, better yet, if R-values decrease when going from Biso to Baniso. Cheers, Ed. What Ed is describing above is sort of what I hoped would happen when I first tried this: the TLS refinement would take care of the global anisotropic atomic motions, and the aniso B refinement would take care of any residual local ones. I realized that using the two together could be unstable, and as several people have pointed out, the data to parameter ratio is pretty low at this resolution to be trying this out. That said, here are the results of the calculations Ed is wondering about above: Starting model: 2096 (non-H) atoms, 249 protein residues, 170 waters. Riding hydrogens built automatically in each refinement cycle. Data set: 23665 unique reflections, 97% complete from 25 - 1.8 A. 5% of reflections taken for free R set. Space group C2, one molecule per asymmetric unit. Refinement run with the same starting model each time, and all parameters except the TLS and B refinement left the same. I set the number of refinement cycles so the refinements would go to convergence: 30 cycles for the non-TLS jobs, 10 TLS + 25 regular for the TLS jobs. One TLS group covering the entire (single-domain) protein. Waters are excluded from TLS refinement, but included in the B refinement. Here are the R and Rfree stats for each job. Biso alone: 0.206 / 0.253 Baniso alone: 0.182 / 0.238 TLS + Biso: 0.188 / 0.228 TLS + Baniso: 0.177 / 0.230 The TLS + Biso job gives the lowest free R and the smallest difference between R and Rfree, so it's the winner. Thanks to all for your input. And thanks to Mischa for answering my original question! - Matt -- Matthew Franklin, Ph. D. Senior Research Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (646) 275-7165
[ccp4bb] refmac problem with anisotropic Us
Hi all - I seem to be encountering a problem with Refmac refinement of a structure containing anisotropic B factors. I have been using TLS refinement and individual anisotropic B refinement, which may not be correct technique, but it does lower my R and Rfree versus aniso B refinement alone (see below). I am asking Refmac to output the TLS contribution to the ANISOU lines, presumably in addition to the aniso B refinement contribution. I also reset the temperature factors to 20 at the beginning of each refinement round. The refinement resolution is 75 to 1.8 A, and the space group is C2, if it matters. The problem comes when I take the output from such a run, model build in Coot, and feed it back in for the next round of refinement. As you can see from the summary table below (trimmed for brevity), the refinement blows up rather badly, making the R factors and the geometry substantially worse. NcycRfactRfree FOM -LL -LLfree rmsBOND zBOND rmsANGL zANGL rmsCHIRAL $$ $$ 0 0.3376 0.3576 0.556115671.6318.1 0.0 0.00.0 0.00.0 5 0.2676 0.2921 0.680109411.6009.2 0.0 0.00.0 0.00.0 9 0.2529 0.2785 0.703108589.5978.4 0.0 0.00.0 0.00.0 10 0.2516 0.2787 0.710108459.5975.2 0.0170 0.829 1.534 0.731 0.097 15 0.2503 0.2876 0.711108711.6042.1 0.0241 0.998 1.729 0.833 0.111 20 0.2739 0.3152 0.672110787.6147.3 0.0268 1.103 1.837 0.885 0.119 25 0.2949 0.3388 0.638112219.6215.4 0.0255 1.049 1.808 0.866 0.116 30 0.3114 0.3575 0.609113094.6257.7 0.0243 1.006 1.794 0.854 0.114 This refinement also throws out an enormous number of warnings about adjacent atoms' B factors being substantially different. Most of these warnings appear to involve the autobuilt riding hydrogens and their adjacent heavy atoms. If I use pdbcur to strip out the ANISOU lines, but otherwise keep the file and refinement protocol unchanged, it goes along nicely: NcycRfactRfree FOM -LL -LLfree rmsBOND zBOND rmsANGL zANGL rmsCHIRAL $$ $$ 0 0.2912 0.3233 0.667112220.6151.2 0.0 0.00.0 0.00.0 5 0.2476 0.2787 0.751107853.5934.7 0.0 0.00.0 0.00.0 9 0.2468 0.2764 0.754107501.5915.6 0.0 0.00.0 0.00.0 10 0.2469 0.2763 0.754107480.5914.2 0.0170 0.829 1.534 0.731 0.097 15 0.1933 0.2387 0.810101501.5723.3 0.0238 0.994 1.791 0.868 0.118 20 0.1849 0.2327 0.817100446.5694.7 0.0239 0.992 1.826 0.884 0.120 25 0.1818 0.2316 0.821100034.5681.0 0.0223 0.925 1.775 0.855 0.114 30 0.1804 0.2296 0.824 99820.5669.4 0.0221 0.913 1.763 0.848 0.113 (Without TLS refinement, the final R and Rfree would be 0.1896 and 0.2503.) So, what's happening here? Does Refmac not like ANISOU lines in the input PDB file? I don't usually work with structures at a resolution high enough to warrant aniso B refinement, so I haven't encountered this before. Thanks for any advice, Matt -- Matthew Franklin, Ph. D. Senior Research Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (646) 275-7165
[ccp4bb] flex-wARP error
Hi all - I'm finding that Arp/Warp is crashing when I try to run a job. I was working on a challenging molecular replacement job where I though Arp tracing the fragmentary density could do a better job than I could by hand (this turned out to be true, by the way). I was testing different parameters, and running a few flex-warp jobs in parallel. They would crash at different cycles of the building and refinement, but always at the same point in the cycle. Looking at the error messages in the log files led to this one, in the last HmainPept log file to be written: Density interpolation: points 10539450 0.00 CPU2 Elapsed Superposition 11394 rotations have been done, total 46821 0.00 CPU1 Elapsed Rotations 2009 flipped peptides are taken end_tag_cputime = data send to stderr: forrtl: severe (64): input conversion error, unit 15, file /Users/matt_franklin/bin/arp_warp_7.1/fort.15 Image PCRoutineLineSource hmain 00106F9F Unknown Unknown Unknown hmain 001063CB Unknown Unknown Unknown hmain 000CCD30 Unknown Unknown Unknown hmain 0009217B Unknown Unknown Unknown hmain 0009192A Unknown Unknown Unknown hmain 000B35C8 Unknown Unknown Unknown hmain 000B0C79 Unknown Unknown Unknown hmain 00012A35 Unknown Unknown Unknown real11.60 user11.51 sys 0.08 I think what's happening here is that I had multiple jobs running simultaneously, and they were all trying to read and write to the same fort.15 file. As you can see, this file is in Arp/Warp's main directory, not in any of the working directories. At some random point, one job would step on the other's read or write, and I'd get a crash. Can someone confirm my guess? Is it possible to fix this bug so that multiple flex-wARP jobs can be run in parallel? I'm running Arp/Warp Expert System (i.e. flex-wARP) from ccp4i, and I don't think this is happening with other Arp/Warp modes. (I'll be happy to provide more logfiles, input files, etc.) Thanks, Matt -- Matthew Franklin, Ph. D. Senior Research Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (646) 275-7165
Re: [ccp4bb] (off-topic) Measurement of channel pore dimensions
On 3/23/11 9:02 AM, Van Den Berg, Bert wrote: Hello all, Does anyone know how to get values for pore sizes of membrane channels? I'm not interested in A x B angstrom values measured between atom centers and assuming a regular pore shape, but a real-life value of either surface area at the narrowest point or the volume of a block centered on the narrowest point of the pore. Thanks, Bert Hi Bert - This is quick and dirty (perhaps too dirty for your purposes), but you could do the following in PyMOL or something similar: - draw the molecular surface of the channel of interest - identify by eye (or from the manuscript) the narrowest point in the pore - place a dummy atom in the middle of the pore at this point - draw a sphere of varying radius using this atom as the center - figure out the largest sphere which still fits inside the channel I would guess that this would give you a number for the radius accurate to 0.5 A, maybe better. It's not a true cross-sectional area, but that doesn't seem as biologically relevant to me as whether a certain sphere (e.g. calcium or magnesium ions) can fit through the pore. Hope that helps, Matt -- Matthew Franklin, Ph. D. Senior Research Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (646) 275-7165
Re: [ccp4bb] Model Building: continuous update of distances as fragment moved
Hi Eric -
I remember that the neighbor atom distances would dynamically update, and
even appear and disappear as your moving atom crosses the 3.2 A distance
threshold to another atom. I don't remember if the regular distance
define distances would do the same; I think not. I'm fairly sure that this
still works - the last time I used O was about six months ago, and the O
package I was using was from c. 2005.
More on-topic, Coot seems to have a dynamic distance command (look under
the Measure menu) which will do what you want.
Hope that helps,
Matt
--
Matthew Franklin, Ph. D.
Senior Research Scientist
New York Structural Biology Center
89 Convent Avenue, New York, NY 10027
(646) 275-7165
_
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Eric
Pettersen
Sent: Tuesday, February 15, 2011 4:14 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Model Building: continuous update of distances as
fragment moved
On Feb 14, 2011, at 4:01 PM, Paul McLaughlin wrote:
In doing some model building I want to move a domain of a protein
manually, as a rigid body, and see in real time continuous updates of
some distances from points on the domain to the rest of the protein (we
have cross linking data).
I dimly remember being able to do with this O,at least a decade ago
(probably more), but it doesn't seem to work in current versions (I
have even dimmer recollections that this might have been a special
feature of a particular graphics card in an SGI)
In any event, does anyone know of anything that might allow us to do
this? { I am not asking about computational ways of exploring alternate
packing of the domain to satisfy distances ( I know about these) -
rather we want to get a feel for what is possible by seeing it for
ourselves).
DIstance monitors in Chimera (www.cgl.ucsf.edu/chimera) automatically update
as structures are moved. Possibly also of interest, Chimera can monitor
steric clashes/contacts as structures are moved:
http://www.cgl.ucsf.edu/chimera/docs/ContributedSoftware/findclash/findclash
.html
--Eric
Eric Pettersen
UCSF Computer Graphics Lab
http://www.cgl.ucsf.edu
Re: [ccp4bb] Crystals that fly around a drop until they land and grow.
Hi Patrick - Just a thought: in a drop that's still equilibrating (i.e. one that's actively nucleating/growing crystals), there will be convection currents, caused by water diffusion out of the drop, protein switching from solution to solid phase, temperature differences, etc. These currents could potentially carry micro-crystals with them until they get too big. Another possibility: crystals are usually denser than their mother liquor, and so tend to end up at the bottom of the drop. The motion seen in the movies could be caused by the micro-crystal nucleating at some random position in the drop, then dropping to the bottom (or sliding down the curved lower face of a hanging drop). I haven't seen this movie myself, so I don't know if this is a reasonable explanation of the motion. - Matt -- Matthew Franklin, Ph. D. Senior Research Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (646) 275-7165 _ From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Patrick Shaw Stewart Sent: Monday, February 14, 2011 5:18 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystals that fly around a drop until they land and grow. A few years ago someone posted on this bb a link to a time-lapse video of crystals growing. There have been a few of these, but in this particular video you could see very small particles that shot across the screen in straight lines or smooth curves before sticking and growing into obvious crystals. They moved rapidly (a minute or so in real time) right across the field of view. The person who posted it commented that they would love to know what was going on with these particles or words to that effect. Does anyone know of or remember a video that shows this effect? I'm asking because this video always puzzled me and a few days ago a possible mechanism occurred to me. Could it be that due to some instability a mini crystal starts to move a little, leaving a trail behind it of depleted protein? Now, is it possible that the higher concentration of protein in front of the crystal causes it to grow faster on that side, so causing it to move forward into the region with higher protein concentration? I have a picture in my mind of a particle being bombarded on all sides by water, precipitant etc, but these particles bounce off again, whereas the protein molecules stick. The momentum picked up by the crystal on each collision is roughly twice the momentum of the particle hitting it in all cases except when a molecule hits and sticks. Then momentum transferred is only one times the momentum of the particle hitting it. The net effect is that the crystal moves in the direction where there is more protein, i.e. forwards. I guess the question is, could the effect be big enough to make a small crystal move more or less in a straight line? I imagine it as a bit like throwing ball bearings at a football on one side, but throwing sticky chewing gum at it (with equal energy) on the other side. The crystal moves around the drop until it becomes so heavy that it stops, a bit like kids rolling snowballs in the park until they can't push them any further. I suggested this mechanism to my colleague here who is a physicist. He was sceptical. Has anyone got any comments? The question has practical implications for crystallization. For example if you add seed crystals to one end of a free interface diffusion experiment (say in a capillary) could micro-crystals buzz up to the far end? Or could a stationary crystal act as a pump, pulling solution past it? Has anything similar been seen with small molecule crystals or crystals in space? Patrick CCP4BB@JISCMAIL.AC.UK - -- patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Looking for the following values...
Hi Jon - - Observed criterion sigma(F) and sigma(I) means what cutoff did your data processing package use to consider a reflection not observed, i.e. throw it out? If you processed your data with Scalepack/HKL2000, the default cutoff is on I, and it's negative 3 sigma. There is no cutoff on F. So you would enter -3 for the sigma(I) entry and leave the sigma(F) entry blank. I don't think that both of these entries are required. - The difference between all reflections and observed reflections is, as far as I can tell, that all reflections indicates the number of mathematically possible unique reflections given your space group, unit cell, and diffraction limits. I generally just leave these entries blank where I can, and where it's required, I divide my number of observed reflections by my percent completeness - for example, a dataset with 31984 unique observed reflections (you can get these numbers from the scaling log files of Scalepack or scala) with 99.8% completeness would give you (31984/0.998)=32048 as the number of all reflections. Hope that helps, Matt -- Matthew Franklin, Ph. D. Senior Research Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (646) 275-7165 _ From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of J. Fleming Sent: Thursday, January 13, 2011 2:49 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Looking for the following values... Hi All, I'm about ready to deposit my structure and have used pdb_extract to aid in the process. Unfortunately the following values were not found and are required by ADIT: 1) Under Data Collection, Reflections section: Observed criterion sigma(F) and Observed criterion sigma(I) 2) Under Refinement, Refinement Statistics section: Number unique reflections (all) I looked in my log files for HKL2000, PHASER, and PHENIX but am confused on where to find the required values above. I tried searching the logs for the mmCIF items but that didn't help. Could someone point me the right direction so I can deposit my structure with the correct values? Thanks in advance, -Jon
Re: [ccp4bb] The density shown in Pymol and Coot is different.
Hi Zhihong - You should use 'FWT' and 'PHWT'. These are the sigma-A weighted 2Fo-Fc map coefficients (likewise, 'DELFWT' and 'PHDELWT' are the sigma-A weighted Fo-Fc map coefficients). The sigma-A weighted 2Fo-Fc map (also known as the mFo-DFc map) has become the standard electron density map to use for model building and for paper figures (unless you're using CNS or Phenix, where you might use a composite-omit version of the same map). Using the 'F' and 'PHIC' coefficients will produce a simple Fo map phased by the model phases; this is generally considered to be more model biased than the sigma-A weighted maps. However, I'm not sure if you're doing the right thing by leaving SIGF unassigned. I believe this should be assigned to the experimental error, which is usually called 'SIGF' in your mtz file. I don't know if FFT actually uses this for anything when it's calculating your map file. I'm also not sure what you should use for the weight parameter - perhaps 'FOM'? I should also point out that you can ask Refmac to output the sigma-A weighted 2Fo-Fc and Fo-Fc maps automatically at the end of a refinement run. If you're using the ccp4i GUI, expand the Monitoring and output options section of the Refmac window, and check the box marked Generate weighted difference maps... Then you don't need to mess around with FFT at all. Hope that helps (and I hope I didn't make any egregious mistakes there!), Matt -- Matthew Franklin, Ph. D. Senior Research Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (646) 275-7165 _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Zhihong Yu Sent: Friday, January 07, 2011 4:31 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] The density shown in Pymol and Coot is different. Hi, Nian, Thanks for your reply. I figured out the problem just now. The difference indeed came from my FFT transformation. I just used all default parameters when I transform A.mtz to A.map, so in the Run FFT dialog, F1= F, Sigma=SIGF, PHI=PHIC, Weight=Unaasigned, and I got A.map under these conditions. Even when I load A.map into Coot, the density is different with A.mtz. But when I assigned parameters as following: F1= FWT, Sigma=Unaasigned, PHI=PHWT, Weight=Unaasigned, and ran the job, I got another A1.map, the density is exactly same as A.mtz this time whatever in Pymol or Coot. I'm a rookie in this field, so herein I have another question about this: Which map should I use for showing the final density, A.map or A1.map? What's the difference of these two maps, it looks like A.map is much stronger than A1.map. Really thanks for response! Zhihong 2011/1/7 Nian Huang huangn...@gmail.com It has been discussed before. Nothing to do with FFT. Both pymol and coot will rescale your map but differently. You have to really look into how the programmer wrote their program to know what is going on. For now, I suggest you just use coot map which is more realistic to me. Also new version of pymol seems to work better. On Fri, Jan 7, 2011 at 1:42 PM, Zhihong Yu nkyuz...@gmail.com wrote: Hi,all I refined a protein-ligand complex structure using Refmac5 and got the map coefficient A.mtz file. I want to represent the electron density in Pymol, so I transformed A.mtz into A.map ccp4-format map file using FFT in ccp4i (parameters are: generate simple map in CCP4 format to cover asymmetric unit, all others are default). When I load A.map inot Pymol and show eletron density around the ligand, I found the electron density was somewhat different comparing to that shown in coot using A.mtz, density in Pymol (using A.map) was much stronger than that in Coot (using A.mtz) when showing at 1.0 sigma level. Where does the differenc come from? Should I set any other parameters when I transform mtz to map using FFT program? Thanks in advance! Zhihong
[ccp4bb] crystallographer position available
To the CCP4BB members: We are seeking to hire a protein crystallographer to work on a new project in our group. Funding is currently available to support the position for one year, with the possibility of renewal at the end of this time. We are looking for someone with 2-5 years of experience beyond their Ph.D. - a newly minted doctorate will likely not have enough experience for the job, while someone with many years of experience in the field would probably not be interested in the salary range we are considering. Also, please note that, as a small non-profit organization, we are unable to provide relocation costs, so applicants from beyond the New York area should keep this in mind. Applications should be directed to me (mfrank...@nysbc.org). The text of the job advertisement follows: The New York Structural Biology Center is a shared research organization, owned and operated by ten universities and medical schools in the New York area, and devoted to the structural analysis of biological processes through the use of NMR, X-ray crystallography, and electron microscopy. We are currently expanding our crystallography lab to begin work on a new project involving the large-scale determination of many protein structures. We are seeking an experienced crystallographer to be an integral part of this work. The successful candidate will work with two other crystallographers and three research assistants, collaborating on all aspects of the structure determination pipeline, from large-scale clone design and construction, to protein expression and purification, to synchrotron data collection, structure determination and PDB submission, to writing research articles describing the significant findings. Applicants should have a Ph.D. in protein crystallography, with 2 - 5 years of postdoctoral experience. A proven track record of multiple successful structure determinations is required. Previous experience with protein expression and purification, and/or cell culture, is highly desirable. Some managerial experience, while not necessary, would be helpful. Salary level is commensurate with qualifications and experience. Thank you for your attention to this. - Matt Franklin -- Matthew Franklin, Ph. D. Senior Research Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (646) 275-7165
Re: [ccp4bb] Extremely long c-axis...reasonable?
Hi Christian - As a number of others have pointed out, this unit cell is unusual, but not impossible. It is, in fact, possible to search the PDB for unit cell parameters. From the home page, choose Advanced Search; on the search page, choose X-ray cell dimensions from the dropdown list of query types. Performing this search finds 12 structures in the PDB with c axis 600 A and a and b axes 100 A. Two of these are fiber diffraction molecular envelopes, but the other 10 are well-refined crystal structures at reasonable resolutions. So it can be done! - Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems, a wholly owned subsidiary of Eli Lilly Company 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Christian Strube Sent: Tuesday, August 03, 2010 7:46 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Extremely long c-axis...reasonable? Hi everybody, indexing of recently collected data gave me unit cell parameters of a=b= 88 and an extremely long c-axis of 652 A. My question is, if the value of the c-axis can be reasonable? Or am I wrong with the SG? I know that length doesn't count, but does anybody has a longer one? Is there a function in the pdb to look for the length of axes? Thanks, Christian Helmholtz-Zentrum f?r Infektionsforschung GmbH | Inhoffenstra?e 7 | 38124 Braunschweig | www.helmholtz-hzi.de Vorsitzende des Aufsichtsrates: MinDir'in B?rbel Brumme-Bothe, Bundesministerium f?r Bildung und Forschung Stellvertreter: MinDirig Heiko Gevers, Nieders?chsisches Ministerium f?r Wissenschaft und Kultur Gesch?ftsf?hrung: Prof. Dr. J?rgen Wehland; Ulf Richter, MBA Gesellschaft mit beschr?nkter Haftung (GmbH) Sitz der Gesellschaft: Braunschweig Handelsregister: Amtsgericht Braunschweig, HRB 477 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
Re: [ccp4bb] pdb-l: Retraction of 12 Structures
After a thorough examination of the available data, which included a re-analysis of each structure alleged to have been fabricated, the committee found a preponderance of evidence that structures 1BEF, 1CMW, 1DF9/2QID, 1G40, 1G44, 1L6L, 2OU1, 1RID, 1Y8E, 2A01, and 2HR0 were more likely than not falsified and/or fabricated and recommended that they be removed from the public record, the university said in its statement this week. I think it's worth pointing out, as I did when this came up last time, that one needs to read this list of PDB codes very carefully, and in a font that distinguishes 0 from O, and 1 from I. It would be very unfortunate, for example, if an innocent bacterial enzyme structure (2HRO) was called into question in some people's minds because of the apparent fabrication of the structure 2HR0. Once again, I'd like to get the community's thoughts: should we ask the PDB to stop using 0 and 1 in its IDs? I'll get off the soapbox now. - Matt (who has nothing to do with any of these structures...) -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems, a wholly owned subsidiary of Eli Lilly Company 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
Re: [ccp4bb] Cold Cabinet Vs Fridge, FPLC
Hi Dianfan - I bought the VWR 2-door chromatography refrigerator (catalog #82009-782) for this purpose and I've been very happy with it. It's currently listed at about 7200 US dollars on VWR's website - much cheaper than 9000 euros, unless the VAT is really that high! If you buy a food grade fridge, you'll get the cooling, but you'll lose important accessories like a floor drain, a hole in the side for computer cables to pass in and out, and a pole in the middle to attach your columns to. One of my colleagues did this, and had to have our facilities department make the modifications later. You can squeeze an entire Akta with fraction collector into a single-door refrigerator (e.g. VWR #55702-520), which is only 4500 USD, but you won't have any space for buffer bottles, fraction racks, spare columns, and the other clutter that usually accumulates around an FPLC. This may not be a bad thing! - Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems, a wholly owned subsidiary of Eli Lilly Company 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Dianfan.Li Sent: Monday, August 31, 2009 5:38 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Cold Cabinet Vs Fridge, FPLC Dear all, Sorry for the non-crystallographic question - I am planing to buy two cold cabinets for the AKTA FPLC/Explorer. They appear to be expensive ( € 9,000 including VAT). Does anybody have experiences using cheaper alternative like food grade fridge to accomodate those machines? If so could I get the vendor information? Thanks in advance, Dianfan __ Dr. Dianfan Li Membrane Structural and Functional Biology Group University of Limerick Limerick, Ireland /HTML Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
[ccp4bb] open research assistant position at ImClone Systems
To anyone who might be interested: I have an opening in my group for a research assistant. The work will consist of cloning and protein expression, primarily using bacteria and insect cells, as well as protein purification and crystallization. I am ideally looking for someone with strong cloning skills and a good laboratory track record. Industrial experience is not necessary but won't hurt either. Please note that this is a junior-level position, suitable for someone with either a bachelor's and 5+ years of relevant experience, or a master's and 2+ years. I will NOT be considering anyone with a Ph.D. for this position. The best way to apply for this job is to go via our company website: http://www.imclone.com/careers.php My position is number 120367. Here's the full job description. GENERAL SUMMARY We are seeking a highly motivated, independent person to work as an essential part of a drug discovery project team. The central duties of this position include cloning, protein expression in bacteria and insect cells, protein purification and biochemical assays. However, in a small group like this one, flexibility is essential since there are many things which need to be done. The candidate must have experience in mammalian or insect cell culture as well as protein expression and purification. The responsibilities of this position can expand to match the desires and abilities of the successful applicant. Candidates should have 3+ years of relevant laboratory experience and at least a BS (MS preferred) in biochemistry or a related discipline. Good oral and written communication skills are absolutely necessary, as is the ability to work with others as a team. Extensive experience in the areas necessary for the job is a plus, but not as important as intelligence and the desire and ability to learn new skills quickly. ESSENTIAL DUTIES AND RESPONSIBILITIES Every effort has been made to identify the essential functions of this position. However, it in no way states or implies that these are the only duties you will be required to perform, nor is it intended to be such a listing of the skills and abilities required to do the job. The omission of specific statements of duties does not exclude them from the position if the work is similar, related, or is an essential function of the position. 1. Designing protein expression constructs for bacterial and baculovirus expression systems 2. Expressing soluble proteins in bacteria and insect cells 3. Purifying proteins on a scale of 5-50 mg using a wide variety of techniques, especially affinity and gel filtration chromatography 4. Protein crystallization, crystal manipulation, and other structural biology work ESSENTIAL KNOWLEDGE, SKILLS, EXPERIENCE 1. Extensive molecular cloning experience 2. Protein purification experience 3. Insect or mammalian cell culture experience 4. The ability to think analytically and work independently 5. Protein crystallization and related skills will be taught on the job -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems, a wholly owned subsidiary of Eli Lilly Company 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
Re: [ccp4bb] Crystallizing Fluorescent Molecules
CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK wrote on 05/14/2009 03:42:05 PM: Dear Crystallographers, I have exactly two spherulite crystals of a protein-peptide complex which have a fluorescently-labelled peptide in them, and are therefore nicely colorful in both the light and fluorescence microscopes, making it easier to know that at least the peptide is in the crystal. However, they are not reproducible. Having gone through the usual list of possible variations which might account for the irreproducibility, I have hit the bottom of the barrel. I was thinking that since the original crystals grew in utter darkness, undisturbed for two weeks while I was away, they were able to nucleate. Is it possible that light exciting the fluorophores is detrimental to crystallization? Or perhaps the complete uniformity of temperature? Even microseeding from one of the spherulites produced nothing (except in the original well.) Any brilliant suggestions welcome... Jacob Keller Hi Jacob - I just want to raise a warning, since a very similar situation bit me back in grad school. I was trying to crystallize RecA with a fluorescently-labeled oligo, and just like you, I got green-glowing crystals, along with some glowing precipitate. I got extremely excited, and a couple of months later had the structure done. There was no DNA in the structure. I had crystallized unliganded RecA, and the fluorescent DNA had precipitated all over the outside of the crystal, painting it and making it look green! Are you certain that your peptide doesn't precipitate under your crystallization conditions? I hope for your sake that my problem is not yours... - Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems, a wholly owned subsidiary of Eli Lilly Company 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
Re: [ccp4bb] Building PEG-400 in Coot
CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK wrote on 04/22/2009 08:12:37 AM: Hi, Could someone point me to the best ways of building a non standard ligand? I had PEG-400 in the cryo, and the density is about twice as long asthe PEG that is in the monomer library. I can get the cif file for PEG-400 from prodrg (1PE). Just wondering what is the best way to use it for importing molecule, real space fitting etc. Attached a photo of the density. Regards, Partha Hi Partha - PEG-400 is a mix of different lengths of polyethylene glycol, but the two dominant species are probably the ones closest in molecular weight to 400 daltons. Those would be octaethylene glycol, with a molecular weight of 370, and nonaethylene glycol, with a molecular weight of 414. Your 1PE molecule is pentaethylene glycol, which has a molecular weight of 238 - I don't know why it's called PEG400. I built a molecule of heptaethylene glycol (MW 326) into a structure of mine that had PEG 400 in the mother liquor (PDB code 1OXN - the ligand is called P33). As I recall, I made an idealized molecule using InsightII, then built it manually into the density using torsion_general in O, plus a great many fiddly adjustments in the molecule's position and orientation. For refinement, I let Refmac auto-generate a suitable dictionary file. You could use my P33 molecule as a starting point for your density. I was only convinced that my tube of density was PEG when I saw the regular pattern of hydrogen bonds alternating with hydrophobic patches that the modeled PEG presented to the protein. Hope that helps, Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems, a wholly owned subsidiary of Eli Lilly Company 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
Re: [ccp4bb] missing carboxyl oxygen
CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK wrote on 03/07/2009 07:11:20 PM: Hi All, Sorry if this is a trivial question. I'm refining my first structure at 2.1 A resolution. There is no density for the main chain carboxyl oxygen of a residue even at 0.5 sigma level in 2FoFc map. The FoFc map shows a negative density when I place the oxygen, satisfying the standard geometry condition. Could someone tell me what to do in this situation. Should I make the occupancy of the oxygen zero. Any comments would be gratefully appreciated. Thanks very much. John Peter Hi John Peter - Is there a positive density peak on the other side of the peptide group? It's quite common to have the peptide bond flipped 180 degrees from the way it should be. The nitrogen atom doesn't move much, so you usually don't see difference density for it, but the oxygen atom moves a good deal and gives difference density peaks. (As others have noted, I'm assuming you meant carbonyl oxygen - the one in a peptide bond - not carboxylate oxygen - the one at the C terminus.) I don't really know about other refinement programs except O, but there should be some sort of flip peptide command - if not, you'll need to move things manually. Just so we're clear, I'm talking about turning this: H Ca-N-C-Ca O into: O Ca-N-C-Ca H Check hydrogen bonding patterns to see if the flipped peptide group makes sense chemically, of course. - Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems, a wholly owned subsidiary of Eli Lilly Company 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
Re: [ccp4bb] protein folds
CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK wrote on 02/25/2009 03:08:10 PM: On Wednesday 25 February 2009 08:20:14 Jayashankar wrote: Dear Folks, The last novel proteins fold were from the yr 2007(pdb statistics), From 2007 to till date no novel fold has been identified, If you reached this conclusion by looking at the PDB web site, you should note that the site explains these numbers are taken from SCOP. The SCOP website states that the most recent update was some time in 2007. So you would have to look elsewhere for new folds deposited since mid-2007. In fact, you have to look no further than SCOP! Or should I say, pre-SCOP, the pre-release partial classification of the latest batch of proteins from the PDB. Here's the webpage: http://www.mrc-lmb.cam.ac.uk/agm/pre-scop/ This says that they're still classifying proteins deposited as recently as Oct 2008, which is a relief to me - I was worried that SCOP had been abandoned. To address the original question, here are some numbers: Folds in SCOP 1.71 (18 Jan 2005): 971 Folds in SCOP 1.73 (26 Sep 2007): 1086 Folds in pre-SCOP (Oct-ish 2008): 1392 We're not done finding new folds yet, folks... - Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
Re: [ccp4bb] temperature after 30 minutes using microscopes ?
CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK wrote on 01/21/2009 05:50:13 PM: Hi there, *warning, reading beyond this line might expose you to non CCP4 related topics* anybody out there who could do the following experiment: Turn on your microscope and measure the temperature after 30 minutes where you would place your precious crystal tray. Okay. Start: 71.9 F Finish: 73.1 F Temperature measured with thermocouple Scotch-taped to the center of the microscope stage, underneath an empty 96-well plate. Room thermostat set to 70. In particular I'm interested in LED versus Halogen driven models. My light source is halogen, but it's coupled to the microscope through a fiber optic light pipe. I know from experience that the older model scopes with the bulb right in the base get quite warm after they've been on for a while. I'm pretty sure my fiber optic setup can be retrofitted to most microscopes... - Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
Re: [ccp4bb] pink/green paper?
CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK wrote on 07/17/2008 11:25:20 AM: Dear all, I have a request for a different kind of paper. Does anyone know of a supplier of the X-ray sensitive pink (or green) paper used to locate X-ray beams. James Hi James - I've used Gafchromic film (www.gafchromic.com) for that purpose. It has better spatial resolution than the pink/green stuff, but works essentially the same way: a spot on the film turns from clear to blue when exposed to X-rays. It won't get fogged by indoor light (although watch out for UV or sunlight) and there's no development needed. Gafchromic sells a whole bunch of films; I'm afraid I don't remember which one you should get. They're rated by radiation dose, but I don't remember how many Grays per second an X-ray generator will deposit in the film... - Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
Re: [ccp4bb] def file for restraining dna and long helices
CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK wrote on 04/10/2008 04:12:23 PM: Sorry for re-posting --this time with subject Hi All, I am sure there are some experienced persons who can help me to check what’s wrong is going with my def file I used in anneal.inp I attached the def and out file. I am trying to restrain the dna (4 chains K, L, D, E) via Watson- Crick base pair and two long helices(A and B) via H-bond in my structure. Any suggestion is well appreciated. Regrards... Raja Hi Raja - You didn't say what problem you're experiencing. From looking at the anneal.out file, it seems that the job finished without any errors, and gave you a pdb file at the end. I suspect the problem you're asking about is that the resulting structure looks terrible - the DNA is all messed up and the base pair hydrogen bonds are broken. And I think I know why. There are two problems that I can diagnose just from looking at the output. First is this: %NOESET-ERR: error in selection - no atoms spec. %NOE-ERR: problem at55 -999.000 -999.000 This error is during the assignment of the Watson-Crick hydrogen bonds. I think you have a few bogus coordinates in your structure; perhaps at atom 55? There are other errors like this. The second, more serious problem can be seen from the starting R factors: R-value by resolution for test set #bin | resolution range | #refl | 1 7.20 500.01143 0.4725 2 5.717.20125 0.5481 3 4.995.71123 0.5232 4 4.544.99123 0.5500 5 4.214.54111 0.5524 6 3.964.21114 0.5904 7 3.763.96109 0.5589 8 3.603.76 84 0.5442 #bin | resolution range | #refl | 1 3.60 500.01932 0.5166 Overall R-value for test set: 0.516556 R-value by resolution for working set #bin | resolution range | #refl | 1 7.20 500.01 1434 0.4355 2 5.717.20 1444 0.5017 3 4.995.71 1410 0.4490 4 4.544.99 1388 0.4777 5 4.214.54 1387 0.4846 6 3.964.21 1175 0.5385 7 3.763.96 1140 0.6060 8 3.603.76 1043 0.5612 #bin | resolution range | #refl | 1 3.60 500.01 10421 0.4748 Overall R-value for working set: 0.474811 You're trying to perform simulated annealing with data that only go to 3.6 A, and that's being generous - based on your R factors, I'd say your model and your data part company at more like 4 A. Annealing simply won't work with such low resolution data. Your ending R factors (43.4 / 53.5) show that all you did was over-refine your structure, causing the free R to increase. Assuming your experimental data only go to 3.6 A, I'm afraid you're going to have a very hard time solving this protein-DNA complex. You definitely can't rely on annealing to help you out - stick with energy minimization and manual rebuilding. If you got your initial model from molecular replacement, you need to be certain that your solution is correct. Sorry for the bad news, Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
Re: [ccp4bb] source for forceps/clamps that fit around cryovials
CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK wrote on 12/07/2007 12:43:55 PM: could anyone drop the name of a source for those tweezer-like clamps whose ends fit perfectly around a Nunc/CryoCap cryovial? one very famous source seems not to have them. these are not the locking clamps or thick metal tongs, but are more like tweezers with the ends curved into half-circles on each arm. -bryan Hi Bryan - They are specimen mount tweezers or SEM mount forceps, originally intended for gripping the groove in the edge of SEM specimen mounts. The old Yale-style cryo pin bases were modified SEM mounts (I think), and the tweezers fit them perfectly. You can buy them from a variety of distributors; they all seem to be reselling Dumont brand products. Here's one website: http://www.emsdiasum.com/microscopy/products/sem/mounts.aspx The tweezers you want are almost at the bottom of the page. HTH, Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
Re: [ccp4bb] X-ray generator uninterruptible power supply
CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK wrote on 03/22/2007 04:46:35 PM: Hi Citizens: Does anyone use an uninterruptible power supply for their X-ray generator? Here at UCDIY, the electricity supply is pretty sketchy, and it is hammering our X-ray generator every time someone forgets to feed the hamster or grease his wheel. If so, how much does such a thing cost, and how unpleasant is it to maintain (since at UCDIY, you get to do all your own maintenance)? Thanks in advance. Bill Hi Bill - I'm afraid a UPS for that kind of power load may be very expensive. My 007HF needs a 20 amp, 200 VAC circuit, so let's say the UPS needs to be able to deliver 4 kW of power (you could probably get away with less). If you're talking about an older power hog like an RU-H3R, that wants an 85 amp (!) 200 VAC circuit, so your UPS needs to handle 17 kW load. Let's assume that you're trying to ride out little glitches (seconds to minutes), not long outages. So you don't need huge battery capacity, just high wattage. Here's a couple of sites that might help you - they helped me when I was looking for a UPS: http://www.powerware.com/UPS/selector/SolutionOverview.asp http://www.apcc.com/template/size/apc/index.cfm? http://www.advizia.com/tripplite/ You'll find that a UPS to run a low-power generator like the 007HF will cost you $5000 - $10,000, while something that can handle an RU-H3R looks like it'll cost $20k and up. I'm not even sure that all of these systems will deliver 3-phase power like the generator needs. I never considered getting a UPS for my generator, even though our building has no backup power and Con Ed also forgets to feed their hamsters occasionally. What I did get was a UPS for my cryostream - I figured that the data collection can always be restarted if the generator died in the middle of the night, but once the crystal is gone, it's gone. I paid less than $10k for a UPS that can run my Cryojet for over 15 hours, so even if the power goes out in the middle of the night, I can come in and rescue the crystal the next day. Email me if you want more info on my choice of UPS for my cryo. - Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
Re: [ccp4bb] Good NMR Board
CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK wrote on 02/08/2007 06:34:44 AM: Thanks for the links to the boards! But I think you could also answer my question. I need a way to remove gas, solved in my protein sample after using pressure filtration. My problem is the small sample volume of 400 mikroliters. greets Justin Hi Justin - I was also going to suggest the MicroCal degasser; of course you have to have the equipment already. Some other, lower-tech possibilities: - Put the sample in a 15-ml Falcon tube. Attach house vacuum to the top of the tube - they can usually take it without cracking. Tap tube vigorously on the benchtop to nucleate the bubbles. - A brief spin in a speedvac might work - just don't leave it in or you'll dry the sample out. - Don't use pressure filtration. Try a small-volume centrifugal filter instead. Like these: http://www.vwrsp.com/catalog/product/index.cgi?catalog_number=82031-362inE=1highlight=82031-362 Disclaimer: I haven't tried the first two methods for this purpose. The first method is similar to how I degassed my calorimetry samples before Microcal introduced its degasser. You might want to try it on a dry tube first... - Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
